Paola Bocchini

Absolute quantitation of lignin pyrolysis products using an internal standard

Abstract A simple method to obtain absolute quantitation of lignin pyrolysis fragments by on-line pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) is proposed. Three compounds were tested as internal standards, i.e. 1,3,5-tri-tert.-butylbenzene, 1,2,4-benzenetricarboxylic acid trimethyl ester and 1,3,5-trimethoxybenzene. The characteristic of the proposed internal standards is that they vaporize in the hot pyrolysis interface during the 3-min equilibration and focus on the top of the GC column, thus avoiding loss of internal standard due to thermal fragmentation. The linearity and reproducibility of response of the standards over a range of concentrations are reported. 1,3,5-Tri-tert.-butylbenzene was selected as the most appropriate among the tested standards to be used in Py–GC–MS of lignin. The correction factors for the main lignin pyrolysis fragments were calculated and a practical application of the proposed method to the analysis of a wheat straw sample is discussed.

Studies on wheat lignin degradation by Pleurotus species using analytical pyrolysis

Abstract The aromatic-ring substitution patterns of p -hydroxyphenyl ( H ), guaiacyl ( G ) and syringyl ( S ) lignin-units are conserved after pyrolytic breakdown enabling analysis of the composition of lignins with different origins. Analytical pyrolysis has been used to investigate lignin degradation by several white-rot fungi including Pleurotus eryngii , a species characterised by the ability to degrade lignin selectively. Using Py-GC/MS it was found that the composition of wheat lignin was modified by P. eryngii causing a decrease of S / G ratio in the residual lignin. Simultaneously, degradation products were incorporated into lignin-polysaccharide complexes. In order to get additional insight on wheat lignin modification by these fungi, the biodegraded samples were permethylated before Py-GC/MS. In this way it was shown that the free phenolic lignin-units (around 50% of the total H and G units and only 6% of S units) were preferentially removed as compared with the etherified ones. Moreover, it was possible to demonstrate that the lignin side-chains were attacked in situ resulting in the increased amounts of ether-linked vanillic and syringic acids, identified as the corresponding methyl esters by Py-GC/MS of permethylated samples. When 50% of wheat lignin was degraded by P. eryngii , the relative abundance of aromatic acids was over 15% of the total lignin derived products. These results validate some of the mechanisms postulated for lignin degradation by white-rot fungi, which were based on simple model compounds.

Pyrolysis-gas chromatography/Mass spectrometry analysis of phenolic and etherified units in natural and industrial lignins

Milled-wood lignin (MWL) from Carpinus betulus, Eucryphia cordifolia, Picea abies, Pinus sp. and Bambusa sp., Kraft lignin from Fagus sylvatica and Eucalyptus globulus, and alkali lignin and hemicellulose-linked lignin from Triticum aestivum, were investigated with respect to their composition in phenolic and etherified phenylpropanoid H (p-hydroxyphenyl), G (guaiacyl) and S (syringyl) units. For this purpose, a methodology based on lignin permethylation, followed by pyrolysis-gas chromatography/mass spectrometry, and quantitation of marker compounds (H-, G- and S-type vinylphenols and their methylated derivatives) in single-ion chromatograms, was developed. The phenolic content in the samples analyzed ranged from 2% of total units in hemicellulose-linked lignin to near 70% in Kraft lignins. Softwood MWL showed higher amounts of phenolic units than MWL from annual plants and hardwoods. It was found that the phenolic content of MWL from the Austral tree species E. cordifolia was unexpectedly high for a hardwood lignin. The significance of this finding in terms of lignin degradability by white-rot fungi, of biotechnological interest, is discussed. Copyright # 1999 John Wiley & Sons, Ltd.

Biotreatment of tannin-rich beer-factory wastewater with white-rot basidiomycete Coriolopsis gallica monitored by pyrolysis/gas chromatography/mass spectrometry

Some fractions of beer-factory wastewaters represent an important environmental concern owing to their high content of polyphenols and dark-brown color. The capacity of Coriolopsis gallica to preferentially degrade lignin has been successfully applied in our laboratory to the biotreatment and decolorization of paper-industry effluents. In this work, the ability of this white-rot fungus to degrade high-tannin-containing wastewaters is evaluated. Under all the conditions studied, effluent decolorization and chemical oxygen demand reduction achieved by C. gallica at day 12 of incubation were close to 50 and 65%, respectively. No adhesion of dark color to the fungal mycelium was observed suggesting that decolorization could be ascribed to C. gallica degradation systems. Mycelium dry-weight values showed that C. gallica is tolerant to relatively high tannin content present in the effluent samples. In the sample containing the highest effluent concentration (60% v/v), dry-weight values suggested an inhibition of fungal growth at day 6 of incubation and a further adaptation of the fungus to the stressing tannin effect at day 12 of fungal treatment. Pyrolysis/gas chromatography/mass spectrometry results showed a decrease of polyphenols pyrolysis products, mainly phenol and guaiacol, with the incubation time. All these results indicate the potential use of C. gallica in bioremediation of tannin-containing industrial wastewaters and in other applications where a reduction in polyphenols content is required.

High-performance liquid chromatographic-electrospray mass spectrometric analysis of phenolic acids and aldehydes

The present work describes the development of an HPLC-electrospray mass spectrometric method for the analysis of phenolic acids and aldehydes. These compounds are important for the quality of foods and feeds, such as dietary fiber supplements, wine and lignicellulose by-products. Good separation was obtained with a phenyl column (3 μm particle size, 150 mm×3.9 mm I.D.), using McOH-H2O (30:70, v/v) as the mobile phase with 0.01% CH,COOH and 0.2 mM tetraethyl ammonium iodide as the ion pairing agent, at a flow-rate of 0.3 ml/min. This system permits post column splitting of the eluate for analysis by electrospray mass spectrometry with a flow-rate of 11 μl/min. This new method is extremely sensitive and less than 6 pg/inj of the studied phenols can be identified and quantified. This method was applied to standard compounds as well as to components of high-fiber dietary supplements (primarily wheat bran), cornmeal, and oat bran.

MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY OF NATURAL AND SYNTHETIC LIGNIN

Lignin is one of the components, with cellulose and hemicellulose, of cell walls in all terrestrial plants. Lignin is an amorphous, three-dimensional, polyphenolic molecule charged with the function of giving strength and impermeability to the plant structure. The determination of its molecular weight and structure is still problematic owing to the difficulties in isolation of lignin from the other cell wall constituents. The present work reports the experimental conditions for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of different types of natural lignins and of one synthetic lignin. A nitrogen laser (λ=337 nm) was used for ion desorption and sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) was chosen as the best matrix for lignin analysis. Data on the molecular weight distribution of oligomers in lignin and information on the monomeric constituents of different types of lignin are shown.

Pyrolysis gas chromatography-mass spectrometry of natural resins used for artistic objects

SummaryEight resins used as protective layers for paintings, small sculptures, jewelries and artistic furniture, namely, Manila copal, colophony, Venice turpentine, elemi, shellac, dammar, sandarac and mastic were subjected to pyrolysis gas chromatography-mass spectrometry (PY-GC-MS). Significant mass spectral ions were monitored in each pyrogram. A table for identification of such resins based on the most significant ions in the mass spectra of the pyrolysis fragments is proposed. The results of PY-GC-MS analysis of a sample from the earth globe Giovanni Enriques, attributed to the Venetian geographer Vincenzo Coronelli (1650–1718) (Museo della Specola, Department of Astronomy, University of Bologna), are reported as an example of practical application of the method to a real artistic sample.

Compositional changes of wheat lignin by a fungal peroxidase analyzed by pyrolysis-GC-MS

Abstract Wheat-straw treatment with a Mn2+-oxidizing peroxidase from the ligninolytic fungus Pleurotus eryngii was investigated using pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Changes in the ratio between p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units were found, consistent with a degradation of S units and increase of the H content after enzymatic treatment. However, the most noticeable modification was the significant decrease of lignin phenolic content as revealed by Py-GC-MS of methylated straw. It was shown that 10 U of peroxidase per mg of straw decreased the amount of phenolic H units from 31% in the control to 3% in the treated straw, that of G units from 40 to 4%, and completely removed the small amount of phenolic S units present in wheat straw. The same tendency to decrease lignin phenolic content was observed when lower enzyme doses were used. These results provide conclusive evidence for modification of lignin during in vitro treatment of a natural lignocellulosic substrate with a ligninolytic peroxidase.

Pyrolysis/gas chromatography/mass spectrometry as a useful technique to evaluate the ligninolytic action of streptomycetes on wheat straw

Abstract Pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS) was used to elucidate whether the chemical modifications produced by streptomycetes on wheat straw are related with the lignocellulose degrading enzymes produced by the microorganism on such substrate. Thus, Streptomyces sp. UAH 47 was grown on wheat straw under solid-state fermentation and periodically the major solubilisation product of this substrate (lignin–carbohydrate complex) and the residual wheat straw were pyrolysed. In parallel, different enzyme activities related with lignocellulose degradation were evaluated. The characteristic pyrolysis products detected by GC/MS from both solubilised lignocarbohydrate complex and residual wheat straw demonstrated an oxidative action of the microorganism on the lignin. Either oxidation or oxidation followed by cleavage of the C3 alkyl chain, from S and G units, mainly at the Cα and Cβ position could be detected. A correlation between the oxidation degree of lignin and both l -3,4-dihydroxyphenylalanine and 2,6-dimethoxyphenol peroxidases produced by the strain can be inferred. Moreover, a liberation of ferulic and p -coumaric acids from the substrate was detected by HPLC, which probably involves the action of ferulic and coumaric acid esterases secreted by the microorganism. These enzymes could be partially responsible for the different pattern in the relative area of 4-vinylguaiacol and 4-vinylphenol detected in all samples along the incubation time. At last, the pyrolysis data suggest a straight correlation between the relative area of the compounds derived from carbohydrates and the pattern of hemicellulolytic and cellulolytic enzymes produced by the strain.

Solid-phase microextraction gas chromatography/mass spectrometric analysis of volatile organic compounds in water

In this work, a solid-phase microextraction (SPME) method for the extraction of volatile organic compounds (VOCs) from water samples has been developed. Fifty-five volatile compounds (from the sixty listed in EPA method no. 524.2) were extracted from aqueous solutions using an SPME fiber coated with Carboxen-polydimethylsiloxane (Carboxen-PDMS), 75 &mgr;m film thickness. Time of sampling and chromatographic separation conditions were optimised. Standard solutions of VOC mixtures with concentrations as low as 0.05 ppb were analysed and their signal/noise ratios measured. Linearity of response for each component of the mixture was tested and mass spectral quality evaluated. A comparison with purge and trap analysis of VOCs was made. The method was applied to real-world samples of drinking, surface and leaching waters. Copyright 1999 John Wiley & Sons, Ltd.